The interaction of cells with their extracellular matrix (ECM) as it occurs in vivo plays a crucial role in the organization, homeostasis, and function of tissues and organs. Continuous communication between cells and their surrounding ECM environment orchestrates critical processes such as the acquisition and maintenance of differentiated phenotypes during embryogenesis, the development of form (morphogenesis), angiogenesis, wound healing, and even tumor metastasis. Both biochemical and biophysical signals from the ECM modulate fundamental cellular activities including adhesion, migration, proliferation, differential gene expression, and programmed cell death.
In turn, the cell can modify its ECM environment by modulating the synthesis and degradation of specific matrix components. The realization of the significance of cell-ECM interaction has led to a renewed interest in characterizing ECM constituents and the basic mechanisms of cell-ECM interaction.
Tissue culture allows the study in vitro of animal cell behavior in an investigator-controlled physiochemical environment. Presumably cultured cells function best (i.e., proliferate and perform their natural in vivo functions) when cultured on substrates that closely mimic their natural environment. Currently, studies in vitro of cellular function are limited by the availability of cell growth substrates that present the appropriate physiological environment for proliferation and development of the cultured cells. Complex scaffolds representing combinations of ECM components in a natural or processed form are commercially available, such as Human Extracellular Matrix (Becton Dickinson) and MATRIGEL®. However, none of the existing scaffolds have been prepared under conditions that regulate the polymerization of the scaffold in a controlled manner so as to produce a composition having mechanical properties and a predetermined 3D microstructure of collagen fibrils and/or soluble ECM components that optimizes cell-substrate interactions to yield predictable and reproducible cellular outcomes. Applicants have discovered that the physical state of an ECM scaffold and not just its molecular composition should be considered in the design of new and improved scaffolds.
As reported herein, modifying the conditions used to form a collagen based matrix from a solubilized collagen solution allows for the controlled alteration of the micro-structural and subsequent mechanical properties of the resulting ECM scaffold. Furthermore, the micro-structural and mechanical properties of the ECM scaffold directly impact fundamental cell behavior including survival, adhesion, proliferation, migration and differentiation of cells cultured within the scaffold.
Basement membrane tissues and submucosal material harvested from warm blooded vertebrates have shown great promise as unique graft materials for inducing the repair of damaged or diseased tissues in vivo, and for supporting fundamental cell behavior (e.g., cell proliferation, growth, maturation, differentiation, migration, adhesion, gene expression, apoptosis and other cell behaviors) of cell populations in vitro. Submucosal material can be extracted or fluidized to provide enriched extracts that can be utilized as additives for tissue culture media, or polymerized to form collagen based scaffolds, to promote in vitro cell growth and proliferation.
As a tissue graft, submucosal tissue undergoes remodeling and induces the growth of endogenous tissues upon implantation into a host. Numerous studies have shown that submucosal tissue is capable of inducing host tissue proliferation, remodeling and regeneration of tissue structures following implantation in a number of in vivo environments, including the urinary tract, the body wall, tendons, ligaments, bone, cardiovascular tissues and other vascular tissues, and the central nervous system. Upon implantation of the submucosal tissues, cellular infiltration and a rapid neovascularization are observed and the submucosa materials are remodeled into host replacement tissue with site-specific structural and functional properties.
Accordingly, submucosa tissue can be used as a tissue graft construct, for example, in its native form, in its fluidized form, in the form of an extract, or as components extracted from submucosa tissue and subsequently purified. The fluidized forms of vertebrate submucosa tissue can be gelled to form a semi-solid composition that can be implanted as a tissue graft construct or utilized as a cell culture substrate. As a tissue graft material, the fluidized form can be injected, or delivered using other methods, to living tissues to enhance tissue remodeling. Furthermore, the fluidized form can be modified, or can be combined with specific proteins, growth factors, drugs, plasmids, vectors, or other therapeutic agents for controlling the enhancement of tissue remodeling at the site of injection. Moreover, the fluidized, solubilized form can be combined with primary cells or cell lines prior to injection to further enhance the remodeling properties that result in the repair or replacement of diseased or damaged tissues.
Because the molecular forces that orchestrate the self assembly of soluble, monomeric collagen into higher ordered structures are weak their assembly can easily turn into an unstructured aggregation of misfolded proteins. In the literature, there are known methods for isolating collagen from a variety of tissues, e.g., placenta and animal tails and using the isolated material to reconstitute collagenous matrices. These known methods rely on the protein's intrinsic ability to retain its secondary structure during protein isolation and assume that, for instance, the alpha helix will retain its helical structure throughout. The end result, even with a homogenous biochemical composition, can be a heterogeneous secondary structure. Controlling the assembly of the constituting monomers into tertiary or quaternary multimeric arrangements is very hard to achieve under such conditions. One embodiment of the present invention is directed to controlling the polymerization of a composition comprising solubilized collagen to form a collagen based scaffold that has the requisite microstructure and composition to allow for the expansion, differentiation and/or clonal isolation of stem cells in a highly reproducible and predictable manner.